Figure 7.
Figure 7. A second assay to measure centriole growth using the centriole distal-end binding protein GFP-Cep97. (A) Micrograph shows a 3D-SIM image of an embryo expressing Asl-GFP (a mother centriole marker), illustrating how mother centrioles in these embryos are preferentially oriented end-on to the cortex (so each mother centriole appears as a hollow ring; see also Fig. S5 and Video 5). Bar, 5 µm. (B) Micrograph shows a 3D-SIM image of a centriole pair in a fixed D. melanogaster spermatocyte expressing the distal-end-binding protein GFP-Cep97. These cells have unusually large centrioles (González et al., 1998), allowing one to easily distinguish the proximal and distal ends. The outer wall of the centriole is revealed by Asl-staining (red), which in spermatocytes is detected on both mother and daughter centrioles. GFP-Cep97 foci (green) are concentrated at the distal end of both the mother (M) and daughter (D) centriole. Bar, 0.5 µm. (C) Schematic shows how the distance between the GFP-Cep97 signal at the distal ends of the mother and daughter would be expected to increase as the centriole grows from early S-phase (d1) to late S-phase (d2). The schematic shows the centriole pair viewed both from the side and the top of the mother. The latter view resembles how the centrioles would usually be viewed in the early embryo, allowing us to measure daughter centriole growth in essentially 2D rather than 3D. Micrographs below show typical images of GFP-Cep97 in early and late S-phase, acquired on a Zeiss-880 Airyscan system. Bar, 0.25 µm. (D) Graph shows daughter centriole length over time, measured using this GFP-Cep97 assay in WT (n = 4 embryos; n = 69 centrioles in gray) and Plk41/2 embryos (n = 5 embryos; n = 84 centrioles in red). These daughter centriole growth profiles are similar to those obtained using the Sas-6-GFP incorporation assay (see Fig. 6 A). Note that centriolar GFP-Cep97 levels decrease toward the end of S-phase, so we cannot accurately track the GFP-Cep97 signal up to NEB. Data are shown as mean ± SEM; R2 is used as a measure for goodness-of-fit.

A second assay to measure centriole growth using the centriole distal-end binding protein GFP-Cep97. (A) Micrograph shows a 3D-SIM image of an embryo expressing Asl-GFP (a mother centriole marker), illustrating how mother centrioles in these embryos are preferentially oriented end-on to the cortex (so each mother centriole appears as a hollow ring; see also Fig. S5 and Video 5). Bar, 5 µm. (B) Micrograph shows a 3D-SIM image of a centriole pair in a fixed D. melanogaster spermatocyte expressing the distal-end-binding protein GFP-Cep97. These cells have unusually large centrioles (González et al., 1998), allowing one to easily distinguish the proximal and distal ends. The outer wall of the centriole is revealed by Asl-staining (red), which in spermatocytes is detected on both mother and daughter centrioles. GFP-Cep97 foci (green) are concentrated at the distal end of both the mother (M) and daughter (D) centriole. Bar, 0.5 µm. (C) Schematic shows how the distance between the GFP-Cep97 signal at the distal ends of the mother and daughter would be expected to increase as the centriole grows from early S-phase (d1) to late S-phase (d2). The schematic shows the centriole pair viewed both from the side and the top of the mother. The latter view resembles how the centrioles would usually be viewed in the early embryo, allowing us to measure daughter centriole growth in essentially 2D rather than 3D. Micrographs below show typical images of GFP-Cep97 in early and late S-phase, acquired on a Zeiss-880 Airyscan system. Bar, 0.25 µm. (D) Graph shows daughter centriole length over time, measured using this GFP-Cep97 assay in WT (n = 4 embryos; n = 69 centrioles in gray) and Plk41/2 embryos (n = 5 embryos; n = 84 centrioles in red). These daughter centriole growth profiles are similar to those obtained using the Sas-6-GFP incorporation assay (see Fig. 6 A). Note that centriolar GFP-Cep97 levels decrease toward the end of S-phase, so we cannot accurately track the GFP-Cep97 signal up to NEB. Data are shown as mean ± SEM; R2 is used as a measure for goodness-of-fit.

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